Aspartate ß-hydroxylase targeting in castration-resistant prostate cancer modulates the NOTCH/HIF1α/GSK3ß crosstalk.

Castration-resistant prostate cancer (CRPC) is an incurable stage of the disease. A multivariate principal component analysis on CRPC in vitro models identified aspartyl (asparaginyl) ß hydrolase (ASPH) as the most relevant molecule associated with the CRPC phenotype. ASPH is overexpressed in various malignant neoplasms and catalyzes the hydroxylation of aspartyl and asparaginyl residues in the epidermal growth factor (EGF)-like domains of proteins like NOTCH receptors and ligands, enhancing cell motility, invasion and metastatic spread. Bioinformatics analyses of ASPH in prostate cancer (PCa) and CRPC datasets indicate that ASPH gene alterations have prognostic value both in PCa and CRPC patients. In CRPC cells, inhibition of ASPH expression obtained through specific small interfering RNA or culturing cells in hypoxic conditions, reduced cell proliferation, invasion and cyclin D1 expression through modulation of the NOTCH signaling. ASPH and HIF1α crosstalk, within a hydroxylation-regulated signaling pathway, might be transiently driven by the oxidative stress evidenced inside CRPC cells. In addition, increased phosphorylation of GSK3ß by ASPH silencing demonstrates that ASPH regulates GSK3ß activity inhibiting its interactions with upstream kinases. These findings demonstrate the critical involvement of ASPH in CRPC development and may represent an attractive molecular target for therapy.

Multifocal Signal Modulation Therapy by Celecoxib: A Strategy for Managing Castration-Resistant Prostate Cancer.

BACKGROUND: Prostate cancer (PCa) is a significant health concern throughout the world. Standard therapy for advanced disease consists of anti-androgens, however, almost all prostate tumors become castration resistant (CRPC). Progression from androgen-sensitive PCa to CRPC is promoted by inflammatory signaling through cyclooxygenase-2 (COX-2) expression and ErbB family receptors/AKT activation, compensating androgen receptor inactivity. METHODS: Making use of CRPC cell lines, we investigated the effects of the anti-inflammatory drug celecoxib. Biochemical data obtained using immunoblotting, enzyme-linked immunosorbent assay (ELISA), invasion, and xenografts were further integrated by bioinformatic analyses. RESULTS: Celecoxib reduced cell growth and induced apoptosis through AKT blockade, cleavage of poly (ADP-ribose) polymerase-1 (PARP-1), and proteasomal degradation of the anti-apoptotic protein Mcl-1. Epidermal growth factor receptor (EGFR), ErbB2, and ErbB3 degradation, and heterogeneous nuclear ribonucleoprotein K (hnRNP K) downregulation, further amplified the inhibition of androgen signaling. Celecoxib reduced the invasive phenotype of CRPC cells by modulating NF-κB activity and reduced tumor growth in mice xenografts when administered in association with the anti-EGFR receptor antibody cetuximab. Bioinformatic analyses on human prostate cancer datasets support the relevance of these pathways in PCa progression. CONCLUSIONS: Signaling nodes at the intersection of pathways implicated in PCa progression are simultaneously modulated by celecoxib treatment. In combination therapies with cetuximab, celecoxib could represent a novel therapeutic strategy to curb signal transduction during CRPC progression.

A hnRNP K⁻AR-Related Signature Reflects Progression toward Castration-Resistant Prostate Cancer.

The major challenge in castration-resistant prostate cancer (CRPC) remains the ability to predict the clinical responses to improve patient selection for appropriate treatments. The finding that androgen deprivation therapy (ADT) induces alterations in the androgen receptor (AR) transcriptional program by AR coregulators activity in a context-dependent manner, offers the opportunity for identifying signatures discriminating different clinical states of prostate cancer (PCa) progression. Gel electrophoretic analyses combined with western blot showed that, in androgen-dependent PCa and CRPC in vitro models, the subcellular distribution of spliced and serine-phosphorylated heterogeneous nuclear ribonucleoprotein K (hnRNP K) isoforms can be associated with different AR activities. Using mass spectrometry and bioinformatic analyses, we showed that the protein sets of androgen-dependent (LNCaP) and ADT-resistant cell lines (PDB and MDB) co-immunoprecipitated with hnRNP K varied depending on the cell type, unravelling a dynamic relationship between hnRNP K and AR during PCa progression to CRPC. By comparing the interactome of LNCaP, PDB, and MDB cell lines, we identified 51 proteins differentially interacting with hnRNP K, among which KLK3, SORD, SPON2, IMPDH2, ACTN4, ATP1B1, HSPB1, and KHDRBS1 were associated with AR and differentially expressed in normal and tumor human prostate tissues. This hnRNP K⁻AR-related signature, associated with androgen sensitivity and PCa progression, may help clinicians to better manage patients with CRPC.

Adaptive phenotype drives resistance to androgen deprivation therapy in prostate cancer.

BACKGROUND: Prostate cancer (PCa), the second most common cancer affecting men worldwide, shows a broad spectrum of biological and clinical behaviour representing the epiphenomenon of an extreme heterogeneity. Androgen deprivation therapy is the mainstay of treatment for advanced forms but after few years the majority of patients progress to castration-resistant prostate cancer (CRPC), a lethal form that poses considerable therapeutic challenges. METHODS: Western blotting, immunocytochemistry, invasion and reporter assays, and in vivo studies were performed to characterize androgen resistant sublines phenotype in comparison to the parental cell line LNCaP. RNA microarray, mass spectrometry, integrative transcriptomic and proteomic differential analysis coupled with GeneOntology and multivariate analyses were applied to identify deregulated genes and proteins involved in CRPC evolution. RESULTS: Treating the androgen-responsive LNCaP cell line for over a year with 10 µM bicalutamide both in the presence and absence of 0.1 nM 5-α-dihydrotestosterone (DHT) we obtained two cell sublines, designated PDB and MDB respectively, presenting several analogies with CRPC. Molecular and functional analyses of PDB and MDB, compared to the parental cell line, showed that both resistant cell lines were PSA low/negative with comparable levels of nuclear androgen receptor devoid of activity due to altered phosphorylation; cell growth and survival were dependent on AKT and p38MAPK activation and PARP-1 overexpression; their malignant phenotype increased both in vitro and in vivo. Performing bioinformatic analyses we highlighted biological processes related to environmental and stress adaptation supporting cell survival and growth. We identified 15 proteins that could direct androgen-resistance acquisition. Eleven out of these 15 proteins were closely related to biological processes involved in PCa progression. CONCLUSIONS: Our models suggest that environmental factors and epigenetic modulation can activate processes of phenotypic adaptation driving drug-resistance. The identified key proteins of these adaptive phenotypes could be eligible targets for innovative therapies as well as molecules of prognostic and predictive value.

Expression of nuclear matrix proteins binding matrix attachment regions in prostate cancer. PARP-1: New player in tumor progression.

Prostate cancer (PCa) displays infrequent point mutations, whereas genomic rearrangements are highly prevalent. In eukaryotes, the genome is compartmentalized into chromatin loop domains by the attachment to the nuclear matrix (NM), and it has been demonstrated that several recombination hot spots are situated at the base of loops. Here, we have characterized the binding between NM proteins and matrix attachment regions (MARs) in PCa. Nontumor and 44 PCa tissues were analyzed. More aggressive tumors were characterized by an increase in the complexity of the NM protein patterns that was synchronous with a decrease in the number of proteins binding the MAR sequences. PARP-1 was the protein that showed the most evident changes. The expression of the PARP-1 associated with NM increased and it was dependent on tumor aggressiveness. Immunohistochemical analysis showed that the protein was significantly overexpressed in tumor cells. To explore the role of PARP-1 in PCa progression, PCa cells were treated with the PARP inhibitor, ABT-888. In androgen-independent PC3 cells, PARP inhibition significantly decreased cell viability, migration, invasion, chromatin loop dimensions and histone acetylation. Collectively, our study provides evidence that MAR-binding proteins are involved in the development and progression of PCa. PARP could play a key role in the compartmentalization of chromatin and in the development of the more aggressive phenotype. Thus, PARP can no longer be viewed only as an enzyme involved in DNA repair, but that its role in chromatin modulation could provide the basis for a new therapeutic approach to the treatment of PCa.

Etoposide and topoisomerase II inhibition for aggressive prostate cancer: Data from a translational study.

BACKGROUND: Etoposide phosphate (VP-16) is a topoisomerase 2 (TOP2) inhibitor that demonstrated activity in patients with metastatic castration-resistant prostate cancer (mCRPC). We investigated the sensitivity of prostate cancer (PCa) cells (LNCaP, 22Rv1, PC3, DU145, PDB and MDB) to VP-16 and the possible relationship between VP-16 activity and TOP2 expression. The activity of VP-16 was compared with that of docetaxel, enzalutamide and olaparib. The prevalence and clinical significance of TOP2 genetic and transcriptomic alterations was also explored in mCRPC. METHODS: Cell cultures and crystal violet cell proliferation assays were performed. Specific antibodies were used in western blots analyses of cell protein extracts. Datasets were analyzed in cBioportal. RESULTS: VP-16 was active in all PCa cell lines analyzed and demonstrated increased activity in PC3 and DU145 cells. VP-16 was more cytotoxic compared to the other treatments, except for LNCaP and 22Rv1, which were more sensitive to docetaxel. Maintenance of antiandrogen treatment in MDB and PDB increased sensitivity to VP-16, docetaxel and enzalutamide. TOP2A was found overexpressed in 22Rv1, DU145 and PC3, whereas TOP2B was overexpressed in 22Rv1 and PDB. In the mCRPC datasets analysis, TOP2A mRNA overexpression was associated with worse patients' prognosis, with the molecular features of neuroendocrine prostate cancer (NEPC) and with lower androgen receptor (AR) score. Patients overexpressing TOP2A mRNA were more likely to harbor RB1 loss. CONCLUSIONS: Specific subpopulations of patients with aggressive variant prostate cancer (AVPC) could benefit from VP-16 treatment. TOP2A overexpression, rather than TOP2B, might be a good biomarker to predict response to VP-16.

Integrative Analysis of Periostin in Primary and Advanced Prostate Cancer.

Periostin (POSTN) is an extracellular matrix protein associated with tumor progression and shorter survival in prostate cancer (PCa). Here, we performed an integrative analysis of POSTN's role in patients with PCa. Clinical and POSTN data from large-scale datasets were analyzed. POSTN cutoffs were identified with X-Tile, and STRING was used for protein-protein interaction analysis. In a cohort of 48 patients with metastatic castration-resistant prostate cancer (mCRPC), we used the AdnaTest platform to isolate circulating tumor cells and extract POSTN mRNA. Plasma samples were also tested for POSTN protein expression by dot blot assay. Data from large-scale datasets did not reveal any association between POSTN genetic alterations and outcome. In primary tumors, we found a significant correlation between POSTN mRNA overexpression, worse baseline prognostic features, and shorter disease-free survival. POSTN was overexpressed in mCRPC and correlated with aggressive features. In our cohort of mCRPC patients, we found a positive correlation between POSTN plasma levels and androgen-receptor variant 7 positivity and an association with shorter overall survival. Our integrative analysis shows that POSTN is associated with poor clinical features and worse outcome in patients with PCa. Further studies are warranted to uncover the function of POSTN in PCa progression and to validate the prognostic significance of POSTN in mCRPC.

Role of Circulating Tumor Cells (CTC), Androgen Receptor Full Length (AR-FL) and Androgen Receptor Splice Variant 7 (AR-V7) in a Prospective Cohort of Castration-Resistant Metastatic Prostate Cancer Patients.

BACKGROUND: Circulating tumor cells (CTC), androgen receptor full-length (AR-FL), and androgen receptor splice variant 7 (AR-V7) are prognostic in patients (pts) with metastatic castration-resistant prostate cancer (mCRPC). AR-V7 seems to predict resistance to androgen-receptor signaling inhibitors (ARSi). METHODS: We assessed the association of CTC, AR-FL, and AR-V7 with prostate-specific antigen (PSA) response and overall survival (OS). We used a modified AdnaTest CTC-based AR-FL and AR-V7 mRNA assay. Chi-square test, Fisher Exact test, Kaplan-Meier method, log-rank test, Cox proportional hazards models were used as appropriate. RESULTS: We enrolled 39 mCRPC pts, of those 24 started a first-line treatment for mCRPC (1L subgroup) and 15 had received at least two lines for mCRPC (>2L subgroup). CTC, AR-FL, and AR-V7 were enriched in >2L compared to 1L subgroup. Detection of these biomarkers was associated with a lower percentage of biochemical responses. Only 1/7 AR-V7+ pts had a PSA response and received cabazitaxel. Median OS was 4.7 months (95% CI 0.6-8.9) in AR-V7+ pts and not reached in AR-V7- pts. AR-V7 was the only variable with prognostic significance in the Cox model. CONCLUSION: AR-V7, CTC, and AR-FL are associated with advanced mCRPC and AR-V7+ predicts for shorter OS.

Interleukin 21 Controls mRNA and MicroRNA Expression in CD40-Activated Chronic Lymphocytic Leukemia Cells.

Several factors support CLL cell survival in the microenvironment. Under different experimental conditions, IL21 can either induce apoptosis or promote CLL cell survival. To investigate mechanisms involved in the effects of IL21, we studied the ability of IL21 to modulate gene and miRNA expressions in CD40-activated CLL cells. IL21 was a major regulator of chemokine production in CLL cells and it modulated the expression of genes involved in cell movement, metabolism, survival and apoptosis. In particular, IL21 down-regulated the expression of the chemokine genes CCL4, CCL3, CCL3L1, CCL17, and CCL2, while it up-regulated the Th1-related CXCL9 and CXCL10. In addition, IL21 down-regulated the expression of genes encoding signaling molecules, such as CD40, DDR1 and PIK3CD. IL21 modulated a similar set of genes in CLL and normal B-cells (e.g. chemokine genes), whereas other genes, including MYC, TNF, E2F1, EGR2 and GAS-6, were regulated only in CLL cells. An integrated analysis of the miRNome and gene expression indicated that several miRNAs were under IL21 control and these could, in turn, influence the expression of potential target genes. We focused on hsa-miR-663b predicted to down-regulate several relevant genes. Transfection of hsa-miR-663b or its specific antagonist showed that this miRNA regulated CCL17, DDR1, PIK3CD and CD40 gene expression. Our data indicated that IL21 modulates the expression of genes mediating the crosstalk between CLL cells and their microenvironment and miRNAs may take part in this process.

Heterogeneous expression and function of IL-21R and susceptibility to IL-21-mediated apoptosis in follicular lymphoma cells.

OBJECTIVE: Interleukin (IL)-21, a member of the IL-2 family, has antitumor activity and is now being tested in non-Hodgkin's lymphoma in combination with anti-CD20 antibodies. IL-21 may either induce apoptosis or promote growth in different lymphoid malignancies. We therefore investigated the IL-21/IL-21R system in follicular lymphoma (FL) cells. MATERIALS AND METHODS: IL-21R expression was studied by reverse transcription polymerase chain reaction, immunofluorescence, and Western blot analyses. Apoptosis was measured by Annexin-V-propidium iodide staining. Signaling via IL-21R was studied using antibodies specific for phosphorylated Janus-activating kinase and signal transducers and activators of transcription proteins by Western Blot. RESULTS: IL-21R was found on primary FL cells in 15 of 15 cases at diagnosis and IL-21 increased apoptosis in 10 of 10 FL samples. However, cells from areas of diffuse growth in FL and from two diffuse lymphomas evolved from previous FL, showed low IL-21R expression. The latter were also resistant to IL-21-mediated apoptosis. Among lymphoma cell lines bearing the t(14;18) translocation, only 1 of 7 showed increased apoptosis in response to IL-21 stimulation. This cell line was IL-21R-positive, whereas five of six nonresponsive cell lines showed very low IL-21R expression. Intriguingly, one of the IL-21-resistant cell lines (DOHH2) expressed high levels of IL-21R. Treatment with IL-21 or IL-4 upregulated suppressor of cytokine signaling 3 gene expression in the IL-21-responsive cell line, but not in DOHH2 cells, which showed defective Janus-activating kinase/signal transducers and activators of transcription signaling in response to IL-21, in relationship to the lack of Janus-activating kinase 3 gene expression. CONCLUSION: These data indicate that low IL-21R expression or defective signal transduction downstream IL-21R may cause refractoriness to IL-21-mediated effects in some FL cells.

The opposite effects of IL-15 and IL-21 on CLL B cells correlate with differential activation of the JAK/STAT and ERK1/2 pathways.

The clonal expansion of chronic lymphocytic leukemia (CLL) cells requires the interaction with the microenvironment and is under the control of several cytokines. Here, we investigated the effect of IL-15 and IL-21, which are closely related to IL-2 and share the usage of the common gamma chain and of its JAK3-associated pathway. We found remarkable differences in the signal transduction pathways activated by these cytokines, which determined different responses in CLL cells. IL-15 caused cell proliferation and prevented apoptosis induced by surface IgM cross-linking. These effects were more evident in cells stimulated via surface CD40, which exhibited increased cell expression of IL-15Ralpha chain and, in some of the cases, also of IL-2Rbeta. IL-21 failed to induce CLL cell proliferation and instead promoted apoptosis. Following cell exposure to IL-15, phosphorylation of STAT5 was predominantly observed, whereas, following stimulation with IL-21, there was predominant STAT1 and STAT3 activation. Moreover, IL-15 but not IL-21 caused an increased phosphorylation of Shc and ERK1/2. Pharmacological inhibition of JAK3 or of MEK, which phosphorylates ERK1/2, efficiently blocked IL-15-induced CLL cell proliferation and the antiapoptotic effect of this cytokine. The knowledge of the signaling pathways regulating CLL cell survival and proliferation may provide new molecular targets for therapeutic intervention.

Interleukin-21 receptor (IL-21R) is up-regulated by CD40 triggering and mediates proapoptotic signals in chronic lymphocytic leukemia B cells.

Interleukin-21 (IL-21) is a member of the IL-2 cytokine family, which mediates proliferation or growth arrest and apoptosis of normal B cells, depending on their activation state. Here we demonstrate that surface IL-21 receptor (R) is expressed at variable levels by chronic lymphocytic leukemia (CLL) B cells freshly isolated from 33 different patients. IL-21R expression was up-regulated following cell stimulation via surface CD40. Therefore, IL-21 effects were more evident in CD40-activated CLL B cells. IL-21 induced an early signaling cascade in CLL B cells, which included JAK-1 and JAK-3 autophosphorylation and tyrosine phosphorylation of STAT-1, STAT-3, and STAT-5. IL-21 signaling failed to stimulate CLL B-cell proliferation, but induced their apoptosis. In addition, IL-21 counteracted the proliferative and antiapoptotic signals delivered by IL-15 to CLL B cells. IL-21-mediated apoptosis involved activation of caspase-8 and caspase-3, cleavage of Bid to its active form t-Bid, and cleavage of PARP and of p27Kip-1. Recent data indicate that CLL B cells require interaction with the microenvironment for their survival and expansion. The present findings thus provide a set of new mechanisms involved in the balance between cell-survival and apoptotic signals in CLL B cells.

CD40 triggering enhances fludarabine-induced apoptosis of chronic lymphocytic leukemia B-cells through autocrine release of tumor necrosis factor-alpha and interferon-gama and tumor necrosis factor receptor-I-II upregulation.

BACKGROUND AND OBJECTIVES: In chronic lymphocytic leukemia (CLL) B-cells are refractory to activation signals and to apoptosis. CD40 triggering, however, rescues CLL B-cells from their anergic state and upregulates the FAS receptor. We therefore studied whether CD40 triggering enhances CLL B-cell sensitivity to fludarabine, and receptors or cytokines potentially involved in apoptosis. DESIGN AND METHODS: CD40-activation of CLL B-cells was carried out by co-culture with CD40L-transfected cells. After fludarabine treatment, apoptosis was evaluated by propidium iodide (PI), annexin-V/PI or DiOC6 staining and flow cytometry analysis. Modulation of Bcl-2, of tumor necrosis factor receptor (TNFRI/II) and release of tumor necrosis factor (TNF)alpha/interferon (IFN)gamma was also analyzed. Furthermore, addition of caspase-inhibitors or anti-TNFalpha/-IFNgamma monoclonal antibodies to fludarabine-treated cells allowed us to determine the mediators of apoptosis. Student's t tests or ANOVA variance statistical analysis were performed to evaluate whether any differences observed might be considered significant. RESULTS: CD40 triggering enhanced fludarabine sensitivity of CLL B-cells, downmodulated Bcl-2 and upregulated TNFRI/II. Caspases 1 and 6 were the major caspases involved in fludarabine apoptosis induction in resting B cells, while only anti-TNFalpha/-IFNgamma monoclonal antibodies reduced apoptosis in activated cells. In agreement with this observation, autocrine production of TNFalpha and IFNgamma by CD40-activated CLL B cells was found. INTERPRETATION AND CONCLUSIONS: B-cells from a considerable proportion of CLL cases studied (11/20) are more prone to fludarabine-induced apoptosis after CD40 triggering; accordingly Bcl-2 expression was lower in activated cells. Moreover, upregulation of TNFRI/II, release of TNFalpha and IFNgamma, and inhibition of apoptosis by anti-TNFalpha/-IFNgamma monoclonal antibodies in CD40-activated cells strongly suggest that these cytokines may play a role in sensitizing B-cells to fludarabine treatment.